An abnormal tumor microenvironment can impair T cell function and limit the immune response. Tumor- derived lactate and an acidic microenvironment, as well as tumor/stromal secretion of TGF can independently and synergistically suppress T cell proliferation, cytokine production and cytotoxicity. We propose to alter components of the tumor microenvironment (pHe, lactate and TGF) and assess these effects on PSMA chimeric antigen receptor (CAR) specific T cell therapy in two highly aggressive, immunocompetent animal models of prostate cancer with orthotopic, lung and bone tumor growth. Our central theme and hypothesis is that a markedly abnormal tumor microenvironment affects T cell tumor-targeting and survival, as well as T cell activation, proliferation and effector-function, and that these T cell functions can be monitored using novel dual reporter systems. A constitutive-reporter will be used for imaging T cell trafficking, tumor targeting and survival, and inducible-reporter systems will concurrently image and assess T cell function. One inducible reporter will monitor T cell activation mediated through a PSMA- specific CAR; the other will monitor TGF-signaling in T cells. The effects of genetically induced abnormalities in the tumor microenvironment (low pHe, high lactate and high TGF) on T cell trafficking and function will be assessed using multimodality imaging techniques. The status of the tumor microenvironment will be assessed repeatedly in the same animal using MRIS to measure pHe and lactate, and microPET to assess tumor hypoxia during tumor growth and during adoptive T cell therapy. Additional ex vivo immunohistochemical cell assays will independently assess the tumor microenvironment and confirm T cell status. We also expect that normalization of different components of the tumor microenvironment (pHe, lactate and TGF) using pharmacological intervention will significantly enhance T cell therapy; therefore, we have included therapeutic sub-aims to test this hypothesis. The relevance and impact of these studies are that: 1) Lactate, pHe, and TGF levels in the tumor microenvironment are frequently abnormal, particularly in more aggressive tumors, and are associated with a reduced immune response. 2) Importantly, there exist specific treatment modalities to normalize these components, and they will be investigated. 3) Many of the imaging strategies developed and used in this project (e.g., MRSI to measure pHe and lactate) could be translated to clinical studies, where monitoring T cell trafficking and function can be monitored in patients using human reporter genes and PET imaging, and where adoptive T cell treatment strategies could be more fully evaluated. PUBLIC HEALTH RELEVANCE: An abnormal microenvironment is characteristic of aggressive prostate cancer, and is associated with a reduced immune response to the tumor; therefore, we propose to study the effects of specific abnormalities in the tumor microenvironment on the immune system. We expect that normalizing these abnormalities in the tumor microenvironment will significantly improve the therapeutic response to immunotherapy. The imaging strategies and therapeutic counter-measures developed in this proposal could be applied to patients with prostate cancer, and could serve as a model for similar studies in other cancers and for stem cell therapy as well.